Strong association of G9a and SUV39h1 with polynucleosomes similar to DNMT3A/3B. (A) Nucleosomes released from nuclei partially digested with MNase at low ionic strength were resolved by ultracentrifugation on a sucrose density gradient (5% to 25%) containing 300 mM NaCl. Gradients were fractionated and analyzed as described in the Methods section. Ponceau S staining shows core histones transferred onto the membrane from the SDS/PAGE gel. The control lanes (denoted as C) on the gels were loaded with unfractionated nuclear extract loaded on the gels to monitor the quality of the immunostaining of the membranes. (B) Quantitation of protein bands obtained from the western blot was done using Quantity One software (Bio-Rad). Plotting of levels of individual proteins in each fraction shows co-sedimentation of G9a and DNMT3A/3B while SUV39h1 shows a sedimentation profile shifted towards bottom of the gradient indicating association with heavier condensed heterochromatin fragments.